Creutzfeldt-Jakob disease (CJD) in humans and scrapie and bovine spongiform encephalopathy (BSE) in animals are some of the diseases that belong to the group of Transmissible Spongiform Encephalopathies (TSE), also known as prion diseases (Prusiner, 1991). These diseases are characterized by an extremely long incubation period, followed by a brief and invariably fatal clinical disease (Roos et al., 1973). To date no therapy is available.
Although these diseases are relatively rare in humans, the risk for the transmissibility of BSE to humans through the food chain has seized the attention of the public health authorities and the scientific community (Soto at al., 2001). Variant CJD (vCJD) is a new disease, which was first described in March 1996 (Will et al., 1996). In contrast to typical cases of sporadic CJD (sCJD), this variant form affects young patients (average age 27 years old) and has a relatively long duration of illness (median 14 months vs. 4.5 months in traditional CJD). A link between vCJD and BSE was first hypothesized because of the association of these two TSEs in place and time (Bruce, 2000). The most recent and powerful evidence comes from studies showing that the transmission characteristics of BSE and vCJD to mice are almost identical and strongly indicating that they are due to the same causative agent (Bruce et al., 1997). Moreover, transgenic mice carrying a human or a bovine gene have now been shown to be susceptible to BSE and vCJD (Scott et al., 1999). Furthermore, no other plausible hypothesis for the occurrence of vCJD has been proposed and intensive CJD surveillance in five European countries, with a low exposure to the BSE agent, has failed to identify any additional cases. In conclusion, the most likely cause of vCJD is exposure to the BSE agent, probably due to dietary contamination with affected bovine central nervous system tissue.
The nature of the transmissible agent has been matter of passionate controversy. Further research, has indicated that the TSE agent differs significantly from viruses and other conventional agents in that it seems not to contain nucleic acids (Prusiner, 1998). Additionally, the physicochemical procedures that inactivate most viruses, such as disrupting nucleic acids, have proved ineffective in decreasing the infectivity of the TSE pathogen. In contrast, the procedures that degrade protein have been found to inactivate the pathogen (Prusiner, 1991). Accordingly, the theory that proposes that the transmissible agent is neither a virus nor other previously known infectious agent, but rather an unconventional agent consisting only of a protein recently gained widespread acceptability (Prusiner, 1998). This new class of pathogen was called a “prion”, short for “proteinaceous infectious particle”. In TSE, prions are composed mainly of a misfolded protein named PrPSc(for scrapie PrP), which is a post-translationally modified version of a normal protein, termed PrPC (Cohen et al., 1998). Chemical differences have not been detected to distinguish these two PrP isoforms and the conversion seems to involve a conformational change whereby the α-helical content of the normal protein diminishes and the amount of β-sheet increases (Pan et al., 1993). The structural changes are followed by alterations in the biochemical properties: PrPC is soluble in non-denaturing detergents, PrPSc is insoluble; PrPC is readily digested by proteases (also called protease sensitive prion protein) while PrPSc is partially resistant, resulting in the formation of a N-terminally truncated fragment known as PrPres is (protease resistant prion protein) (Cohen et al., 1998).
The notion that endogenous PrPC is involved in the development of infection is supported by experiments in which endogenous PrP gene was knocked out where the animals were both resistant to prion disease and unable to generate new infectious particles (Bueler et al., 1993). In addition, it is clear that during the tune between the inoculation with the infectious protein and the appearance of the clinical symptoms, there is a dramatic increase in the amount of PrPSc.
These findings suggest that endogenous PrPC is converted to PrPSc conformation by the action of an infectious form of the PrP molecule (Soto et al., 2001). Prion replication is hypothesized to occur when PrPSc in the infecting inoculum interacts specifically with host PrPC, catalyzing its conversion to the pathogenic form of the protein. A physical association between the two isoforms during the infectious process is suggested by the primary sequence specificity in prion transmission (Telling et al., 1994) and by the reported in vitro generation of PrPSc-like molecules by mixing purified PrPC with PrPSc (Saborio et al., 2001). However, the exact mechanism underlying the conversion is not known.
Investigations with chimeric transgenes showed that PrPSc and PrPC are likely to interact within a central domain delimited by codons 96 and 169 (Prusiner, 1996) and synthetic PrP peptides spanning the region 109-141 proved to be able to bind to PrPC and compete with PrPSc interaction (Chabry et al., 1998).
Based on data with transgenic animals, it has been proposed that additional brain factors present in the host are essential for prion propagation (Telling et al., 1995). It has been demonstrated previously that prion conversion does not occur under experimental conditions where purified PrPC and PrPSc are mixed and incubated (Saborio et al., 1999) but that the conversion activity is recovered when the bulk of cellular proteins are added back to the sample (Saborio et al., 1999). This finding provides direct evidence that other factors present in the brain are essential to catalyse prion propagation.
The observation that cholesterol depletion decreases the formation of PrPSc whereas sphingolipid depletion increases PrPSc formation, suggested that “lipid rafts”(lipid domains in membranes that contain sphingolipids and cholesterol) may be the site of the PrPc to PrPSc conversion reaction involving either a raft-associated protein or selected raft lipids (Fantini et al., 2002). However, the role of lipid rafts in prion infectivity is still unclear.
Several in vitro methods of detections of prions in a sample have been developed. The set of known detection methods, include PrPSc detection methods using specific ligand carriers selected from aminoglycans, fibronectin and Apolipoprotein A (WO 02/065133); methods using the monoclonal antibodies selected from Gö138, 3B5 and 12F10 (Schulz et al., 2000); methods based on the formation of a complex between PrPSc and Apolipoprotein H (WO 03/005037); or methods based on the PrPSc in vitro amplification called protein misfolding cyclic amplification (PMCA) described in Saborio et al., 2001 and Lucassen et al., 2003.
Apolipoprotein B is the major protein component of the two known atherogenic lipoproteins, Low Density Lipoproteins (LDL) and remnants of triglyceride-rich lipoproteins. The apolipoprotein B concentration is considered to be a direct reflection of the number of atherogenic particles in the blood and has been proposed as a parameter for determining the risk of atherosclerosis.
Apolipoprotein E is a constituent of several plasma lipoprotein such as chylomicrons, very low-density lipoproteins (VLDL), and high-density lipoproteins (HDL) (Lehninger et al., 1993).
Apolipoprotein E has recently emerged as a major genetic risk factor for Alzheimer's disease, a neurodegenerative disorder (U.S. Pat. No. 6,022,683) and upregulated in the cerebrospinal fluid of patients with variant CJD and Alzheimer's disease compared to patients with sporadic CJD (Choe et al., 2002). The Apolipoprotein E 4/4 phenotype is associated with increased risk of coronary heart diseases and Creutzfeld-Jakob disease (Golaz et al., 1995). Apolipoprotein E gene expression was found to be increased in astrocytes associated with the neuropathological lesions in a scrapie animal model (Dietrich et al., 1991).
Apolipoprotein E was found to recognise a shared structural motif of amyloids and prion which, after induction, can accelerate the adoption of a beta-sheet conformation (Baumann et al., 2000).
Apolipoprotein B and E are ligands for the LDL receptor and are known for its prominent role in cholesterol transport and plasma lipoprotein metabolism via LDL receptor interactions (Segrest et al, 2001; Clavey et al, 1991).
One approach to the treatment and prevention of prion diseases has been to develop agents for blocking the transformation of PrPc into PrPSc. Some agents proposed were Congo red dye (U.S. Pat. No. 5,276,059), nerve growth peptides (U.S. Pat. No. 5,134,121), fragments of prion proteins (U.S. Pat. No. 6,355,610), compounds that reduces Apolipoprotein E release in the brain tissue (US 2002/0155426), therapeutic agents that prevent Apolipoprotein E4 to interact with neuronal LDL receptor-related protein (WO 97/14437), compounds that increase Apolipoprotein E levels (WO 99/15159) and beta-sheet breaker peptides (U.S. Pat. No. 5,948,763).
It would be desirable to develop new methods for identifying and inhibiting the prion conversion factor(s).